Transgenic plants expressing insecticidal proteins and methods of producing the same

ABSTRACT

The invention provides DNA sequences encoding insecticidal secreted proteins from bacteria and transgenic plants having insecticidal activity against coleopteran insect pests. Methods of protecting plants from damage by insects by transforming the plants with chimeric genes comprising DNA sequences encoding the insecticidal secreted proteins are also provided.

This application is a continuation divisional of application Ser. No.09/858,525, filed on May 17, 2001 now U.S. Pat. No. 6,706,860, which isa continuation of 09/573,872, filed May 18, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new insecticidal secreted proteins(“ISPs”) isolated from a bacterial strain, preferably a Brevibacillusspecies strain, most preferably a Brevibacillus laterosporus speciesstrain which are insecticidal when ingested in combination with anISP-complimentary protein such as another ISP protein of this invention,and to DNA sequences encoding such proteins. These proteins are usefulto prevent or minimize insect damage, particularly of corn rootworms, toplants in a field.

The present invention also relates to plants, particularly corn plants,which are rendered insecticidal, preferably to coleopteran insects,particularly to Diabrotica spp., Leptinotarsa spp. and Anthonomusspecies insects, by the expression of the ISP proteins of this inventionin cells of said plants.

The present invention also relates to a method for controlling damage byDiabrotica spp., Leptinotarsa spp. or Anthonomus species insects,preferably Diabrotica spp. insect pests, particularly corn rootworms, byhaving the ISP proteins of the invention, particularly the proteins withthe amino acid sequence of any one of SEQ ID No. 2, 4, 8 or 10, orinsecticidally-effective fragments thereof, ingested by said insects.

2. Description of the Prior Art

Some of the most destructive pests are found among the Diabroticinebeetles. In North America, the three important species of cornrootworms, Diabrotica virgifera (the Western corn rootworm), Diabroticabarberi (the Northern corn rootworm) and Diabrotica undecimpunctatahowardi (the Southern corn rootworm) are considered to be the mostexpensive insect pests to control (Metcalf, 1986, Foreword in “Methodsfor the Study of Pest Diabrotica”, pp. vii–xv, eds. Krysan, J. L. andMiller, T. A., Springer-Verlag, New York). Diabrotica virgifera andDiabrotica barberi are considered the most serious insect pests of cornin the major corn-producing states of the United States and Canada(Levine and Oloumi-Sadeghi, 1991, Annu. Rev. Entomol. 36, 229–55). Thelarvae feed on the roots and thus cause direct damage to corn growth andcorn yields. Costs for soil insecticides to control larval damage to theroot systems of corn and aerial sprays to reduce beetle damage to cornsilks, when combined with crop losses, can approach one billion dollarsannually (Metcalf, 1986, supra). Recently, in some US states it wasdiscovered that the crop rotation program of planting soybeans aftercorn lost its effect as corn rootworms have adapted to this situation.

Bacterial strains and/or genes with toxicity to corn rootworm have beendescribed in U.S. Pat. Nos. 6,023,013; 6,015,553; 6,001,637; 5,906,818;and 5,645,831. Also, PCT publications WO 00/09697, WO 99/57282, WO98/18932, WO 97/40162, and WO 00/26378 relate to toxins and genesobtainable from Bacillus or other bacterial spp., some of which aredescribed to have toxicity to corn rootworm. WO 98/44137, WO 94/21795and WO 96/10083 relate to pesticidal Bacillus strains, characterized bypesticidal proteins and auxiliary proteins produced during vegetativegrowth, some of which are described to have toxicity to corn rootworm.U.S. Pat. No. 5,055,293 describes a method to control corn rootworms byinoculating soil with parasporal-inclusion forming species of Bacilluslaterosporus. Orlova et al. (1998, Applied Environmental Microbiol. 64,2723) showed insecticidal activity to mosquitoes associated with proteincrystals in crystal-forming strains of Bacillus laterosporus.

OBJECTS AND SUMMARY OF THE INVENTION

An object of the present invention is to provide novel proteins and DNAsequences encoding such proteins with significant toxicity to insects,preferably Diabrotica spp. insects, particularly corn rootworm.

In one embodiment of the invention, a protein is provided comprising theamino acid sequence of the smallest active toxin of the protein of SEQID No. 2, wherein said smallest active toxin is:

-   a) a fragment of the protein of SEQ ID No. 2, and-   b) insecticidal to Diabrotica virgifera larvae when ingested by said    larvae in combination with the protein of SEQ ID No. 4 from amino    acid position 51 to 457. Also provided is a protein comprising the    amino acid sequence of the smallest active toxin of the protein of    SEQ ID No. 4, wherein said smallest active toxin is:-   a) a fragment of the protein of SEQ ID No. 4, and-   b) insecticidal to Diabrotica virgifera larvae when ingested by said    insect in combination with the protein of SEQ ID No. 2 from amino    acid position 38 to 871.

Particularly preferred is a protein characterized by an amino acidsequence comprising the sequence of SEQ ID No. 2 from amino acidposition 38 to amino acid position 768 or 781, preferably the proteincharacterized by the amino acid sequence of SEQ ID No. 2 or SEQ ID No.10; and a protein characterized by an amino acid sequence comprising thesequence of SEQ ID No. 4 from amino acid position 51 to amino acidposition 449 or 457, preferably a protein characterized by the aminoacid sequence of SEQ ID No. 4 or SEQ ID No. 8.

A further object of the invention is a protein comprising the amino acidsequence of the protease-digestion fragment of the protein encoded bythe isp1A DNA deposited at the BCCM-LMBP under accession number LMBP4009, which protease-digestion fragment is insecticidal to Diabroticavirgifera upon combined application with the protein of SEQ ID. No. 4from amino acid position 51 to amino acid position 457; and a proteincomprising the amino acid sequence of the protease-digestion fragment ofthe protein encoded by the isp2A DNA deposited at the BCCM-LMBP underaccession number LMBP 4009, which protease-digestion fragment isinsecticidal to Diabrotica virgifera upon combined application with theprotein of SEQ ID No. 2 from amino acid position 38 to amino acidposition 871; particularly wherein said protease-digestion fragment isobtainable by treatment with coleopteran gut juice.

Also provided in accordance with this invention is a DNA sequenceencoding the above proteins, particularly a DNA comprising an artificialDNA sequence having a different codon usage compared to the naturallyoccurring DNA sequence but encoding the same protein sequence,preferably contained in a chimeric gene operably linked to aplant-expressible promoter region (i.e., a promoter region which issuitable for expression in plant cells, this can be from bacterial,viral or plant origin or can be artificially made); particularly apromoter region which is preferentially active in root tissue.

In one embodiment of the invention, the promoter in said chimeric genecomprises the DNA sequence of SEQ ID No. 5 or 6 or a DNA hybridizingthereto under stringent hybridization conditions.

In a further embodiment of this invention, the chimeric gene furthercomprises a signal peptide for secretion from the cell or for targetingto a cellular organelle, particularly a chloroplast transit peptide.

Also provided is a plant cell, a plant or a seed, comprising any ofthese chimeric genes integrated in their cells, particularly acombination of the chimeric gene encoding the ISP1A, or aninsecticidally effective fragment thereof, and the chimeric geneencoding the ISP2A protein, or an insecticidally effective fragmentthereof; particularly a corn cell, plant or seed.

In another embodiment of this invention, a micro-organism transformed tocontain any of the above DNA sequences is provided.

Also provided is a process for controlling insects, particularly aprocess for rendering a plant resistant to coleopteran insects,comprising expressing any of the ISP proteins of this invention in cellsof a plant, and regenerating transformed plants from said cells whichare resistant to insects. In such process, the insect is preferablyselected from the group consisting of: rootworms, weevils, potatobeetles, Diabrotica species, Anthonomus spp., Leptinotarsa spp.,Agelastica alni, Hypera postica, Hypera brunneipennis, Halticatombacina, Anthonomus grandis, Tenebrio molitor, Triboleum castaneum,Dicladispa armigera, Trichispa serica, Oulema oryzae, Colaspis brunnea,Lissorhorptrus oryzophilus, Phyllotreta cruciferae, Phyllotretastriolata, Psylliodes punctulata, Entomoscelis americana, Meligethesaeneus, Ceutorynchus sp., Psylliodes chrysocephala, Phyllotretaundulata, Leptinotarsa decemlineata, Diabrotica undecimpunctataundecimpunctata, Diabrotica undecimpunctata howardi, Diabrotica barberi,and Diabrotica virgifera.

Yet another object of the present invention is to provide a method forrendering plants insecticidal against Coleoptera and a method forcontrolling Coleoptera, comprising planting, sowing or growing in afield plants transformed with DNA sequences encoding the ISP proteins ofthe invention, particularly corn plants. In an embodiment of thisinvention, the ISP proteins are combined with other insecticidalproteins or protein combinations, preferably corn rootworm-toxicproteins.

Also provided in accordance with this invention are ISP1A or ISP2Aequivalents, preferably from a Brevibacillus laterosporus strain,particularly from a Brevibacillus laterosporus strain not formingcrystalline inclusions. Such equivalents preferably have molecularweights of about 95 to about 100 kD for ISP1A equivalents, and about 45to about 50 kD for ISP2A equivalents, as determined in standard 8–10%SDS-PAGE gel electrophoresis using appropriate molecular weight markers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

In accordance with this invention, new bacterial toxins and DNAsequences encoding them have been isolated and characterized. The newproteins were designated ISP1A and ISP2A, and the DNA sequences encodingthem isp1A and isp2A.

In accordance with this invention “ISP1A protein” refers to any proteincomprising the smallest protein fragment of the amino acid sequence ofSEQ ID No. 2 which retains insecticidal activity, particularly tocoleopteran insects, more particularly to corn rootworm, cotton bollweevil and Colorado potato beetle, preferably to Diabrotica spp.,especially to Southern, Western and Northern corn rootworm, particularlyto Diabrotica virgifera, upon combined ingestion by an insect with asuitable ISP-complimentary protein, particularly the mature ISP2Aprotein. This includes any protein with an amino acid sequencecomprising the amino acid sequence from the amino acid at position 32 or38, preferably 38, to an amino acid at a position from amino acidposition 768 to amino acid position 871 in SEQ ID No. 2, particularlyany protein with an amino acid sequence comprising at least the aminoacid sequence of SEQ ID No. 2 from amino acid position 38 to amino acidposition 768. This also includes the protein obtained from the aminoacid sequence of SEQ ID No. 2 by cleaving off part or all of theN-terminal signal peptide sequence or the protein wherein the signalpeptide sequence has been replaced by another, e.g. a plant, targetingpeptide, a methionine amino acid or a methionine-alanine dipeptide.Specifically, this includes the protein comprising an N-terminalprokaryotic or eucaryotic, e.g. bacterial or plant, signal peptide forsecretion or targeting. This also includes hybrids or chimeric proteinscomprising the above described smallest toxic protein fragment, e.g., ahybrid between an ISP1A and ISP2A protein of this invention. Further,included in the designation “ISP1A”, as used herein, are alsoprotease-resistant fragments of the ISP1A protein retaining insecticidalactivity obtainable by treatment with insect gut juice, preferablycoleopteran gut juice, particularly coleopteran gut proteases,preferably corn rootworm proteases, e.g., cysteine proteinases, serineproteinases, trypsin, chymotrypsin or trypsin-like proteases.Particularly, the coleopteran is selected from the group consisting of:corn rootworm, cotton boll weevil and Colorado potato beetle, Diabroticaspp., Diabrotica virgifera, Diabrotica barberi, Diabroticaundecimpuncata, Leptinotarsa decemlineata, and Anthonomus grandis. In apreferred embodiment of this invention, an ISP1A protein according tothis invention is not insecticidal when ingested in isolation withoutproviding simultaneously or sequentially, an ISP complimentary proteinsuch as an ISP2A protein.

In accordance with this invention “ISP2A protein” refers to any proteincomprising the smallest protein fragment of the amino acid sequence ofSEQ ID No. 4 which retains insecticidal activity, particularly tocoleopteran insects, more particularly to corn rootworm, cotton bollweevil and Colorado potato beetle, preferably to Diabrotica spp.,especially to Diabrotica virgifera, Diabrotica barberi, Diabroticaundecimpuncata, upon combined ingestion by an insect with a suitableISP-complimentary protein, particularly the mature ISP1A protein. Thisincludes any protein with an amino acid sequence comprising the aminoacid sequence from the amino acid at position 43 or 51, preferably 51,to the amino acid at a position from amino acid position 449 to position457 in SEQ ID No. 4, particularly any protein with an amino acidsequence comprising at least the amino acid sequence of SEQ ID No. 4from amino acid position 51 to position 449. This also includes theprotein obtained from the amino acid sequence of SEQ ID No. 4 bycleaving off part or all of the N-terminal signal peptide sequence ofthe protein, and the protein wherein the signal peptide has beenreplaced by another, e.g., a plant, targeting peptide, a methionineamino acid or a methionine-alanine dipeptide. Specifically, thisincludes the protein comprising an N-terminal prokaryotic or eucaryotic,e.g. bacterial or plant, signal peptide for secretion or targeting. Thisalso includes hybrids or chimeric proteins comprising the abovedescribed smallest toxic protein fragment, e.g., a hybrid between anISP1A and ISP2A protein of this invention. Further, included in thedesignation “ISP2A”, as used herein, are also protease-resistantfragments of the ISP2A protein retaining insecticidal activityobtainable by treatment with insect gut juice, preferably coleopterangut juice, particularly coleopteran gut proteases, e.g., cysteineproteinases, serine proteinases, trypsin, chymotrypsin or trypsin-likeproteases. In a preferred embodiment of this invention, an ISP2A proteinaccording to this invention is not insecticidal when ingested inisolation without providing simultaneously or sequentially, an ISPcomplimentary protein such as an ISP1A protein.

An “ISP-complimentary protein”, as used herein, refers to a protein,including but not limited to the mature ISP1A or ISP2A protein, which incombination with one of the ISP proteins of this invention, isinsecticidal upon ingestion by an insect, particularly a coleopteraninsect, preferably a corn rootworm, a cotton boll weevil or Coloradopotato beetle, more particularly Diabrotica virgifera, Diabroticabarberi, Diabrotica undecimpuncata or Anthonomus grandis. Particularly,also VIP (“Vegetative Insecticidal Protein”) proteins and activefragments thereof, particularly the mature VIP proteins with theirsignal sequences cleaved off, as described in WO 98/44137, WO 94/21795and WO 96/10083 are ISP-complimentary proteins in accordance with thisinvention. An ISP-complimentary protein to the ISP1A protein is ideallythe ISP2A protein or the VIP2Aa or VIP2Ab protein or active fragmentsthereof (such as the mature proteins with the signal sequences removed)as described in U.S. Pat. No. 5,990,383, or any bacterial secretedprotein which has a sequence identity of at least 50%, preferably atleast 75%, particularly at least 85%, to any one of the ISP2 or VIP2proteins, and which is insecticidal when ingested by an insect,preferably a coleopteran insect, particularly a corn rootworm, incombination with the mature ISP1A protein. An ISP-complimentary proteinto the ISP2A protein is ideally the mature ISP1A protein, the VIP1Aa orVIP1Ab protein or active fragments thereof (such as the mature proteinwith the signal peptide removed) as described in U.S. Pat. No.5,990,383, or any bacterial secreted protein which has a sequenceidentity of at least 50%, preferably at least 75%, particularly at least85%, to any one of the ISP1 or VIP1 proteins, and which is insecticidalwhen ingested by an insect, preferably a coleopteran insect,particularly a corn rootworm, in combination with the mature ISP2Aprotein. For the avoidance of doubt, an ISP-complimentary protein and anISP protein are always different proteins.

In a preferred embodiment of this invention, the ISP proteins of thisinvention, or their equivalents, when used in isolation, i.e., withoutany of the complementary ISP proteins present, do not result in anysignificant insecticidal activity, preferably to corn rootworm larvae,particularly to Diabrotica virgifera, when tested in a surfacecontamination assay on standard insect diet, and this at a concentrationwherein the proteins (each applied in that concentration) in combinationresult in 100% mortality, preferably to corn rootworm larvae,particularly to Diabrotica virgifera. Particularly, the ISP1 proteins ofthis invention give no significant mortality (i.e., no difference withthe controls using a buffer solution alone) to Diabrotica virgiferalarvae when only an ISP1 protein is applied at a concentration of 70ng/cm2 in a surface contamination assay using standard corn rootwormdiet, and the ISP2 proteins of this invention give no significantmortality (i.e., no difference with the controls using a buffer solutionalone) to Diabrotica virgifera larvae when only an ISP2 protein isapplied at a concentration of 36 ng/cm2 in a surface contamination assayusing standard corn rootworm diet, while ISP1 and ISP2 proteins appliedtogether in these concentrations in the same type of assay give 100%mortality to these larvae.

As used herein, “ISP1A equivalent” or “ISP2A equivalent” refers to aprotein with the same or substantially the same toxicity to a targetinsect as the ISP1A or ISP2A protein, respectively, when applied to suchtarget insect, preferably when ingested by such insect, in a binarycombination with an ISP-complimentary protein, and with substantiallythe same amino acid sequence as the ISP1A or ISP2A protein,respectively. Also included in the definition of ISP1A or ISP2Aequivalents are bacterial proteins of respectively about 45 to about 50kD and about 95 to about 100 kD molecular weight as determined bystandard 8–10% SDS-PAGE gel electrophoresis, preferably from aBrevibacillus laterosporus strain, particularly from a Brevibacilluslaterosporus strain not forming crystalline inclusions, which proteinsin combination but not in isolation, have significant insecticidalactivity to corn rootworm larvae.

The use of the terms “in combination”, when referring to the applicationof an ISP protein (or its equivalent) and an ISP-complimentary proteinto a target insect to get an insecticidal effect, includes thesimultaneous application (i.e., in the same feed, cells or tissue andapplied or ingested by an insect at the same moment) and the separate,sequential application (i.e. one is provided after the other but notapplied or ingested at the same time) of an ISP protein (or itsequivalent) and an ISP-complimentary protein of this invention, as longas these proteins are found together in the insect gut at one moment intime. The ISP1A protein of this invention could thus be expressed in acertain type of cells or a certain zone in the roots of a plant, whilethe ISP2A protein of this invention could be expressed in another kindof cells or another zone in the roots of the same plant, so that theproteins will only interact once root material is ingested. Alsoincluded herein is the expression of an ISP protein or its equivalent inroots of a plant, particularly a corn plant, and the expression of anISP-complimentary protein in root-associated bacteria such asrhizobacteria strains (or vice versa).

“The same toxicity to a target insect”, with respect to an ISP proteinand an ISP equivalent protein as used herein, means that the meanmortality of the ISP protein, in the presence of a suitableISP-complimentary protein, is not significantly different from the meanmortality of the ISP equivalent, also in the presence of the samesuitable ISP-complimentary protein. Particularly, this refers to thesituation wherein the 95% fiducial limits of the LC50 of the ISP protein(when tested in the presence of a suitable ISP-complimentary protein)overlap with the 95% fiducial limits of the LC50 of the ISP equivalent(when tested in the presence of a suitable ISP-complimentary protein).

“Substantially the same toxicity to a target insect”, as used hereinwith respect to an ISP protein and an ISP equivalent protein, refers tolevels of mean mortality of such proteins to a target insect which aresignificantly different between the ISP and the ISP equivalent protein,but which are still within a range of insecticidal activity which isuseful to control or kill the relevant target insect, preferably whensuch protein is expressed in a plant. In a preferred embodiment of thisinvention, proteins have substantially the same toxicity to a targetinsect when their LC50 values for such a target insect in the same(replicated) in vitro assay conducted under the same assay conditionsdiffer from each other by a factor 2 to 100, preferably 2 to 50,particularly 2 to 20, most preferably 2 to 10.

Also, functionally analogous amino acids can be used to replace certainamino acids in an ISP1A or ISP2A protein to obtain ISP1A or ISP2Aequivalents. For example, one or more amino acids within the sequencecan be substituted by other amino acids of a similar polarity which actas a functional equivalent, resulting in a silent alteration withrespect to functionality of the protein. Substitutes for an amino acidwithin the sequence can be selected from other members of the class towhich the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Proteins wherein conservativeamino acids replacements are made using amino acids of the aboveindicated same class which retain substantially the same insecticidalactivity to a target insect compared to the original protein, areincluded herein as equivalents of the ISP proteins of this invention.

A protein with “substantially the same amino acid sequence” to an ISP1Aprotein, as used herein, refers to a protein with at least 90%,particularly at least 95%, preferably at least 97% sequence identitywith the ISP1A protein, wherein the percentage sequence identity isdetermined by using the blosum62 scoring matrix in the GAP program ofthe Wisconsin package of GCG (Madison, Wis., USA) version 10.0 (GCGdefaults used). “Sequence identity”, as used throughout thisapplication, when related to proteins, refers to the percentage ofidentical amino acids using this specified analysis. The “sequenceidentity”, as used herein, when related to DNA sequences, is determinedby using the nwsgapdna scoring matrix in the GAP program of theWisconsin package of GCG (Madison, Wis., USA) version 10.0 (GCG defaultsused).

“ISP protein” or “ISP protein of this invention”, as used herein, refersto any one of the new proteins of this invention and identified hereinas ISP1A or ISP2A protein or their equivalents. An “ISP protoxin” refersto the full length ISP1A or ISP2A protein as it is encoded by thenaturally-occurring bacterial DNA sequence, including the signalpeptide. An “ISP toxin” refers to an insecticidal fragment thereof,particularly the smallest toxic fragment thereof or the mature proteinwith the signal peptide removed. A “mature” ISP, as used herein, refersto the ISP protein of this invention as secreted by its native bacterialhost cell, which is insecticidally active in combination with anISP-complimentary protein (without the N-terminal bacterial signalpeptide sequence). A mature ISP protein can have a complete nativeC-terminal or can have a C-terminal truncation.

Also included in this invention as an ISP protein is a first proteinwith an apparent molecular weight of its mature form of about 95 toabout 100 kD, particularly about 100 kD, which is secreted by abacterium, preferably a bacterium which is not B. thuringiensis or B.cereus, particularly a bacterium which is Brevibacillus laterosporus, orinsecticidally effective fragments of said first protein, characterizedby a significant insecticidal activity when combined with a secondprotein with an apparent molecular weight of its mature form of about 45to about 50 kD, preferably about 45 kD, which is secreted by abacterium, preferably a bacterium which is not B. thuringiensis or B.cereus, particularly a bacterium which is Brevibacillus laterosporus, orinsecticidally effective fragments of said second protein, wherein thecombination of said first and said second protein, or their insecticidalfragments, is significantly insecticidal to larvae of the Coloradopotato beetle, the Western corn rootworm, the Southern corn rootworm,and the Northern corn rootworm, when ingested by said insects, andwherein said protein combination is not significantly insecticidal toOstrinia nubilalis, Spodoptera frugiperda, Heliothis virescens,Helicoverpa zea and Sesamia nonagroides, when ingested by said insects,and wherein said first protein alone has no significant insecticidalactivity when ingested by an insect. Also included herein is the secondprotein as described above which is insecticidal when ingested by aninsect in combination with the above-described first protein.

“Apparent molecular weight”, as used herein, is the molecular weight asevidenced by SDS-PAGE analysis upon comparison with molecular weightstandards. As is appreciated by the skilled person, this molecularweight is an approximate determination and can have a range of variationof about 10–15% with respect to the actual molecular weight asdetermined based on the amino acid sequence.

As used herein, the terms “isp1A DNA” or “isp2A DNA” refer to any DNAsequence encoding the ISP1A or ISP2A protein, respectively, as definedabove. This includes naturally occurring, artificial or synthetic DNAsequences encoding the newly isolated proteins or their fragments asdefined above, particularly DNA sequences with a modified codon usageadapted to more closely match the codon usage of a plant. Examples ofartificial DNA sequences encoding an ISP1A and ISP2A protein are shownin SEQ ID Nos. 9 and 7, respectively (the ISP proteins encoded by theseDNA sequences are defined herein as ISP1A-1 and ISP2A-1). Also includedherein are DNA sequences encoding insecticidal proteins which aresimilar enough to the DNA sequence of SEQ ID No. 1, 3, 7 or 9 so thatthey can (i.e., have the ability to) hybridize to these DNA sequencesunder stringent hybridization conditions. “Stringent hybridizationconditions”, as used herein, refers particularly to the followingconditions wherein hybridization is still obtained: immobilizing a firstDNA sequence on filters, and prehybridizing the filters for either 1 to2 hours in 50% formamide, 5% SSPE, 2× Denhardt's reagent and 0.1% SDS at42° C. or 1 to 2 hours in 6×SSC, 2×Denhardt's reagent and 0.1% SDS at68° C. The denatured radiolabeled second DNA is then added directly tothe prehybridization fluid and incubation is carried out for 16 to 24hours at one of the above selected temperatures. After incubation, thefilters are then washed for 20 minutes at room temperature in 1×SSC,0.1% SDS, followed by three washes of 20 minutes each at 68° C. in0.2×SSC and 0.1% SDS. An autoradiagraph is established by exposing thefilters for 24 to 48 hours to X-ray film (Kodak XAR-2 or equivalent) at−70° C. with an intensifying screen. Of course, equivalent conditionsand parameters can be used in this process while still retaining thedesired stringent hybridization conditions. As used herein, stringenthybridization preferably occurs between DNA sequences with at least 90to 95%, preferably at least 97%, particularly 99%, sequence identity.

Also included in the definition of “isp1 DNA” are all DNA sequencesencoding a protein with a sequence identity of at least 90%, preferablyat least 95%, particularly at least 97%, most preferably at least 99%with the protein of SEQ ID. No. 2 and which has substantially the same,preferably the same, insecticidal activity of the protein of SEQ ID No.2, wherein said protein sequence identity is determined by using theblosum62 scoring matrix in the GAP program of the Wisconsin package ofGCG (Madison, Wis., USA) version 10.0 (GCG defaults used). Included inthe definition of “isp2 DNA” are all DNA sequences encoding a proteinwith a sequence identity of at least 90%, preferably at least 95%,particularly at least 97%, most preferably at least 99%, with theprotein of SEQ ID. No. 4 and which has substantially the same,preferably the same, insecticidal activity of the protein of SEQ ID No.4, wherein said protein sequence identity is determined by using theblosum62 scoring matrix in the GAP program of the Wisconsin package ofGCG (Madison, Wis., USA) version 10.0 (GCG defaults used).

An “isp gene” or “isp DNA”, as used herein, is a DNA sequence encodingan ISP protein in accordance with this invention, referring to any oneof the isp1A or isp2A DNA sequences defined above.

An “isp DNA equivalent” or an “isp gene equivalent”, as used herein, isa DNA encoding an ISP equivalent protein as defined above.

The terms “DNA/protein comprising the sequence X” and “DNA/protein withthe sequence comprising sequence X”, as used herein, refer to a DNA orprotein including or containing at least the sequence X in theirnucleotide or amino acid sequence, so that other nucleotide or aminoacid sequences can be included at the 5′ (or N-terminal) and/or 3′ (orC-terminal) end, e.g., an N-terminal transit or signal peptide. The term“comprising”, as used herein, is open-ended language in the meaning of“including”, meaning that other elements then those specifically recitedcan also be present. The term “consisting of”, as used herein, isclosed-ended language, i.e., only those elements specifically recitedare present. The term “DNA encoding a protein comprising sequence X”, asused herein, refers to a DNA comprising a coding sequence which aftertranscription and translation results in a protein containing at leastamino acid sequence X. A DNA encoding a protein need not be anaturally-occurring DNA, and can be a semi-synthetic, fully synthetic orartificial DNA and can include introns and 5′ and/or 3′ flankingregions. The term “nucleotide sequence”, as used herein, refers to thesequence of a DNA or RNA molecule, which can be in single- ordouble-stranded form.

The term “gene”, as used herein refers to a DNA coding region flanked by5′ and/or 3′ regulatory sequences allowing an RNA to be transcribedwhich can be translated to a protein, typically comprising at least apromoter region. A “chimeric gene”, when referring to an isp DNA of thisinvention, refers to an isp DNA sequence having 5′ and/or 3′ regulatorysequences different from the naturally-occurring bacterial 5′ and/or 3′regulatory sequences which drive the expression of the ISP protein inits native host cell.

“Insecticidal activity” or “insecticidally effective”, when referring toan ISP protein of the invention or its equivalents, as used herein,means the capacity of an ISP protein to kill insects above the levelsfound in control treatment under the same assay conditions, upon theingestion of such protein by an insect, preferably a coleopteran insect,particularly a corn rootworm, especially Diabrotica virgifera, incombination with an ISP-complimentary protein, such as a second ISPprotein. Preferably, the second ISP protein is the other protein encodedby the same bacterial operon as the first ISP or aninsecticidally-effective fragment or equivalent thereof, yieldingoptimal insect mortality upon ingestion of the combined proteins.“Insect-controlling amounts” of an ISP protein, as used herein, refersto an amount of ISP protein which is sufficient to limit damage on aplant by insects feeding on such plant to commercially acceptablelevels, e.g. by killing the insects or by inhibiting the insectdevelopment or growth in such a manner that they provide less damage toa plant and plant yield is not significantly adversely affected, whensuch ISP protein is provided with an ISP-complimentary protein, such asanother ISP protein, preferably the other protein encoded by the sameoperon as the first ISP, or an insecticidally effective fragmentthereof. “Insecticidally-effective ISP fragment”, as used herein, refersto a fragment of an ISP protein of this invention which retainsinsecticidal activity when provided in combination with anISP-complimentary protein, such as another ISP protein, preferably theother ISP encoded by the same operon as the first ISP. In the abovedefinitions related to insecticidal activity, the insect preferably is alarva in any of the larval stages. Throughout this application,reference can be made to an “isp DNA and insecticidally-effectivefragments or equivalents thereof”, and in that case the insecticidalactivity obviously refers to the activity of the protein encoded by theDNA and not to the insecticidal activity of the DNA itself.

In accordance with this invention, the ISP proteins of this inventionand their equivalents, particularly the mature ISP1A and ISP2A proteins,were found to have no significant insecticidal activity to lepidopteraninsects selected from the group consisting of: Heliothis virescens,Helicoverpa zea, Manduca sexta, Helicoverpa armigera, Spodopteralifforalis, Spodoptera frugiperda, Sesamia nonagroides, and Ostrinianubilalis.

In accordance with this invention, target insects susceptible to the ISPproteins of the invention or their equivalents are contacted with theseproteins in insect-controlling amounts, preferably insecticidal amounts,by expression in a transgenic plant of DNA sequences encoding such ISPproteins. Said target insects will only be affected by the insecticidalproteins when they ingest plant tissue. Thus, in another object of thepresent invention a method is provided for rendering plants insecticidalagainst Coleoptera and a method for controlling Coleoptera, comprisingplanting, sowing or growing in a field plants transformed with DNAsequences encoding the ISP proteins of the invention, particularly cornplants.

The signal peptide of the ISP proteins of the invention can be removedor modified according to procedures known in the art, see, e.g.,published PCT patent application WO 96/10083, or they can be replaced byanother peptide such as a chloroplast transit peptide (e.g., Van DenBroeck et al., 1985, Nature 313, 358, or preferably the modifiedchloroplast transit peptide of U.S. Pat. No. 5,510,471) causingtransport of the protein to the chloroplasts, by a secretory signalpeptide or a peptide targeting the protein to other plastids,mitochondria, the ER, or another organelle, or it can be replaced by amethionine amino acid or by a methionine-alanine dipeptide. Signalsequences for targeting to intracellular organelles or for secretionoutside the plant cell or to the cell wall are found in naturallytargeted or secreted proteins, preferably those described by Klösgen etal. (1989, Mol. Gen. Genet. 217,155–161), Klösgen and Weil (1991, Mol.Gen. Genet. 225, 297–304), Neuhaus & Rogers (1998, Plant Mol. Biol. 38,127–144), Bih et al. (1999, J. Biol. Chem. 274, 22884–22894), Morris etal. (1999, Biochem. Biophys. Res. Commun. 255, 328–333), Hesse et al.(1989, EMBO J. 8 2453–2461), Tavladoraki et al. (1998, FEBS Lett. 426,62–66), Terashima et al. (1999, Appl. Microbiol. Biotechnol. 52,516–523), Park et al. (1997, J. Biol. Chem. 272, 6876–6881), Shcherbanet al. (1995, Proc. Natl. Acad. Sci USA 92, 9245–9249), all of which areincorporated herein by reference, particularly the signal peptidesequences from targeted or secreted proteins of corn. Although a DNAsequence encoding such a plant signal peptide can be inserted in thechimeric gene encoding the ISP1A and in the chimeric gene encoding theISP2A protein for expression in plants, in one embodiment of thisinvention, a DNA sequence encoding such a signal peptide is onlyinserted in the chimeric ISP2A gene and the ISP1A chimeric gene lacks asignal peptide, or has a methionine amino acid or a methionine-alaninedipeptide instead of the signal peptide. In a preferred embodiment ofthis invention, the proteins are secreted from the roots as described byGleba et al. (1999, Proc. Natl. Acad. Sci. USA 25, 5973–5977) andBorisjuk et al., 1999 (Nat. Biotechn. 17, 466–469), using a strongroot-preferred promoter, particularly a strong root-preferred promoterfrom corn, and an N-terminal signal peptide for secretion from the rootcells, preferably from corn, particularly the signal peptide of asecreted protein, preferably a corn secreted protein, selected from thegroup of: expansin, alpha-amylase, polyamine oxidase, cytokinin oxidase,endoxylanase.

Plants included in the scope of this invention are corn, cotton,soybean, peas, beans, lentils, potato, tomato, tobacco, lettuce,Brassica species plants, sugarcane, rice, oilseed rape, mustard,asparagus, wheat, barley, coffee, tea, vines, rubber plant, beet, turfgrasses, sorghum, oats, rye, onions, carrots, leek, cucumber, squash,melon, sunflower, particularly any plant which is susceptible to damageby coleopteran insects, preferably corn rootworm, potato beetles orweevils, particularly insects of the Diabrotica or Leptinotarsa species,most preferably any insect selected from the group consisting of:Diabrotica virgifera, Diabrotica barberi, Diabrotica undecimpuncata,Leptinotarsa decemlineata and Anthonomus grandis, most preferably cornplants.

“Corn”, as used herein, refers to all plants of the species Zea mays,and any seeds, roots or grain, or other materials containing or directlyproduced from corn cells, of any variety of Zea mays, including but notlimited to field corn, sweet corn, hybrid corn, white corn and dentcorn, whether hybrid or inbred lines. Preferably, the corn plants usedin this invention are suitable parent lines for producing hybrid corn,and most preferably they already carry an endogenous or transgenicinsect-resistance giving them protection from the major cornlepidopteran insect pests including but not limited to Ostrinianubilalis.

The ISP1A and ISP2A proteins of this invention can be isolated in aconventional manner from the E. coli strain, deposited under theprovisions of the Budapest Treaty on Jan. 11, 2000 at the BCCM-LMBP(Belgian Coordinated Collections of Microorganisms—Laboratorium voorMoleculaire Biologie—Plasmidencollectie, University of Gent, K. L.Ledeganckstraat 35, B-9000 Gent, Belgium) under accession number LMBP4009, or more preferably, they can be isolated from the supernatant of aBacillus strain, preferably a crystal-minus Bacillus thuringiensisstrain, transformed with a plasmid containing the approximately 7 kbXbaI-EcoRI fragment of the plasmid pUCIB120/ISP as deposited underaccession number LMBP 4009. The ISP proteins can be used to preparespecific monoclonal or polyclonal antibodies in a conventional manner(Höfte et al., 1988, Appl. Environm. Microbiol. 54, 2010). The ISPproteins can be treated with a protease such as cysteine proteases, toobtain the protease-resistant fragments of the ISP proteins.

The DNA sequences encoding the ISP proteins can be isolated in aconventional manner from the deposited strain or can be synthesizedbased on the encoded amino acid sequence.

DNA sequences encoding other ISP proteins in accordance with thisinvention can be identified by digesting total DNA from isolatedbacterial strains with restriction enzymes; size fractionating the DNAfragments, so produced, into DNA fractions of 5 to 10 Kb, preferably 7to 10 Kb; ligating these fractions to cloning vectors; screening the E.coli, transformed with the cloning vectors, with a DNA probe that wasconstructed from a region of known ISP protein genes or with a DNA probebased on specific PCR fragments generated from isp DNA using primerscorresponding to certain regions within known ISP protein genes. Such“other ISP proteins”isolated in accordance with this invention, like theISP proteins of this invention, are characterized by any or all,preferably all, of the following characteristics: a) their significantinsecticidal activity to larvae of the Southern corn rootworm,Diabrotica undecimpunctata, and to larvae of the Colorado potato beetle,Leptinotarsa decemlineata, upon ingestion of such other ISP protein withan ISP-complimentary protein, preferably the mature ISP1A or ISP2Aprotein of this invention; b) their lack of significant insecticidalactivity to larvae of lepidopteran insects, preferably Ostrinianubilalis, upon ingestion of such other ISP protein with anISP-complimentary protein, preferably the mature ISP1A or ISP2A proteinsof this invention; c) their apparent molecular weight of about 100 kD orabout 45 kD of the mature forms (without signal peptide), d) theiroccurrence in the supernatant of a bacterial strain culture which is notBacillus thuringiensis or Bacillus cereus, and e) by their lack ofsignificant toxicity to corn rootworms, preferably Diabrotica virgifera,upon ingestion in the absence of an ISP complimentary protein.

Of course, any other DNA sequence differing in its codon usage butencoding the same protein or a similar protein with substantially thesame insecticidal activity, can be constructed, depending on theparticular purpose. It has been described in some prokaryotic andeucaryotic expression systems that changing the codon usage to that ofthe host cell is desired for gene expression in foreign hosts (Bennetzen& Hall, 1982, J. Biol. Chem. 257, 3026; Itakura, 1977, Science 198,1056–1063). Codon usage tables are available in the literature (Wada etal., 1990, Nucl. Acids Res. 18, 2367–1411; Murray et al., 1989, NucleicAcids Research 17, 477–498) and in the major DNA sequence databases.Accordingly, synthetic DNA sequences can be constructed so that the sameor substantially the same proteins are produced. It is evident thatseveral DNA sequences can be devised once the amino acid sequence of theISP proteins of this invention is known. Such other DNA sequencesinclude synthetic or semi-synthetic DNA sequences that have been changedin order to inactivate certain sites in the gene, e.g. by selectivelyinactivating certain cryptic regulatory or processing elements presentin the native sequence, or by adapting the overall codon usage to thatof a more related host organism, preferably that of the host organism inwhich expression is desired. Synthetic DNA sequences could also be madefollowing the procedures described in EP 0 385 962, EP 0 618 967, or EP0 682 115.

Small modifications to a DNA sequence such as described above can beroutinely made by PCR-mediated mutagenesis (Ho et al., 1989, Gene 77,51–59; White et al., 1989, Trends in Genet. 5, 185–189). New syntheticor semi-synthetic genes can be made by automated DNA synthesis andligation of the resulting DNA fragments.

To prevent or delay the development of resistance by coleopteraninsects, particularly corn rootworm, cotton boll weevil or Coloradopotato beetle, preferably Leptinotarsa decemlineata, Diabrotica barberi,Diabrotica undecimpuncata, Anthonomus grandis or Diabrotica virgifera,to transgenic hosts, particularly plants, expressing ISP proteins ofthis invention or their equivalents, it is preferred to also express inthe same host, preferably a transgenic plant, another protein or anotherprotein complex, which has a different mode of action, and a hightoxicity to the same insect targeted by the first toxin or toxin complexwhen produced in a transgenic host, preferably a plant. Suitablecandidates to be combined with the ISP1A and ISP2A of the inventioninclude the mature VIP1Aa protein when combined with the mature VIP2Aaor VIP2Ab protein of PCT publication WO 96/10083 in case these VIPproteins have a different mode of action compared to the ISP proteins;the corn rootworm toxins of Photorhabdus or Xenorhabdus spp., e.g., theinsecticidal proteins of Photorhabdus luminescens W-14 (Guo et al.,1999, J. Biol. Chem. 274, 9836–9842); the CryET70 protein of WO00/26378; the insecticidal proteins produced by Bt strains PS80JJ1,PS149B1 and PS167H2 as described in WO 97/40162, particularly the about14 kD and about 44 kD proteins of Bt strain PS149B1; the Cry3Bb proteinof U.S. Pat. No. 6,023,013; protease inhibitors such as the N2 and R1cysteine proteinase inhibitors of soybean (Zhao et al., 1996, PlantPhysiol. 111, 1299–1306) or oryzastatine such as rice cystatin (Genbankentry S49967), corn cystatin (Genbank entries D38130, D10622, D63342)such as the corn cystatin expressed in plants as described by Irie etal. (1996, Plant Mol. Biol. 30, 149–157). Also included herein are allequivalents and variants, such as truncated proteins retaininginsecticidal activity, of any of the above proteins.

Such combined expression can be achieved by transformation of a plantalready transformed to express a corn rootworm toxic protein or proteincomplex, or by crossing plants transformed to express different cornrootworm toxic proteins. Alternatively, expression of the ISP proteinsof the invention can be induced in roots upon feeding of corn rootwormlarvae on root tissue, e.g., by using a wound-induced promoter region,preferably a wound-induced root-preferred promoter region.

The 5 to 10, preferably 7 to 10, Kb fragments, prepared from total DNAof the isp genes of the invention, can be ligated in suitable expressionvectors and transformed in E. coli, and the clones can then be screenedby conventional colony immunoprobing methods (French et al., 1986,Anal.Biochem. 156, 417–423) for expression of the toxin with monoclonalor polyclonal antibodies raised against the ISP proteins. Also, the 5 to10 Kb fragments, prepared from total DNA of the bacterial strains of theinvention, can be ligated in suitable Bt shuttle vectors (Lereclus etal., 1992, Bio/Technology 10, 418) and transformed in a crystal minusBt-mutant. The clones are then screened for production of ISP proteins(by SDS-PAGE, Western blot and/or insect assay).

The genes encoding the ISP proteins of this invention can be sequencedin a conventional manner (Maxam and Gilbert, 1980, Methods in Enzymol.65, 499–560; Sanger, 1977, Proc. Natl. Acad. Sci. USA 74, 5463–5467) toobtain the DNA sequence. Sequence comparisons indicated that the genesare different from previously described genes encoding proteins secretedduring the vegetative growth phase of Bacillus or other bacterialspecies and Bacillus thuringiensis crystal proteins with activityagainst Coleoptera (Crickmore, et al., 1998, Microbiology and MolecularBiology Reviews Vol 62: 807–813; WO 98/44137, WO 94/21795, WO 96/10083,WO 00/09697, WO 9957282, and WO 9746105).

In order to express all or an insecticidally effective part of the DNAsequence encoding an ISP protein of this invention in E. coli, in otherbacterial strains or in plants, suitable restriction sites can beintroduced, flanking the DNA sequence. This can be done by site-directedmutagenesis, using well-known procedures (e.g., Stanssens et al., 1989,Nucleic Acids Research 12, 4441–4454; White et al., 1989, supra). Forexpression in plants, the isp DNA of the invention, or its equivalent,is usually contained in a chimeric gene, flanked by 5′ and 3′ regulatorysequences including a plant-expressible promoter region and 3′transcription termination and polyadenylation sequence active in plantcells. In order to obtain improved expression in plants, the codon usageof the isp DNA or its equivalent of this invention can be modified toform an equivalent, modified or artificial gene or gene part, or the ispDNA or insecticidally-effective parts thereof can be inserted in thechloroplast genome and expressed there using a chloropast-activepromoter (e.g., Mc Bride et al., 1995, Bio/Technology 13, 362). Forobtaining enhanced expression in monocot plants such as corn, a monocotintron also can be added to the chimeric gene, and the isp DNA sequenceor its insecticidal part can be further changed in a translationallyneutral manner, to modify possibly inhibiting DNA sequences present inthe gene part by means of site-directed intron insertion and/or byintroducing changes to the codon usage, e.g., adapting the codon usageto that most preferred by the specific plant (Murray et al., 1989,supra) without changing significantly the encoded amino acid sequence.

The insecticidally effective isp DNA or its equivalent, preferably theisp chimeric gene, encoding an ISP protein, can be stably inserted in aconventional manner into the nuclear genome of a single plant cell, andthe so-transformed plant cell can be used in a conventional manner toproduce a transformed plant that is insect-resistant. In this regard, adisarmed Ti-plasmid, containing the insecticidally effective isp genepart, in Agrobacterium tumefaciens can be used to transform the plantcell, and thereafter, a transformed plant can be regenerated from thetransformed plant cell using the procedures described, for example, inEP 0 116 718, EP 0 270 822, PCT publication WO 84/02913 and publishedEuropean Patent application (“EP”) 0 242 246 and in Gould et al. (1991,Plant Physiol. 95, 426–434). Preferred Ti-plasmid vectors each containthe insecticidally effective isp chimeric gene sequence between theborder sequences, or at least located to the left of the right bordersequence, of the T-DNA of the Ti-plasmid. Of course, other types ofvectors can be used to transform the plant cell, using procedures suchas direct gene transfer (as described, for example in EP 0 233 247),pollen mediated transformation (as described, for example in EP 0 270356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611), plantRNA virus-mediated transformation (as described, for example in EP 0 067553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (asdescribed, for example in U.S. Pat. No. 4,536,475), and other methodssuch as the methods for transforming certain lines of corn (Fromm etal., 1990, Bio/Technology 8, 833–839; Gordon-Kamm et al., 1990, ThePlant Cell 2, 603–618, U.S. Pat. No. 5,767,367) and rice (Shimamoto etal., 1989, Nature 338, 274–276; Datta et al., 1990, Bio/Technology 8,736–740) and the recently described method for transforming monocotsgenerally (PCT publication WO 92/09696).

Different conventional procedures can be followed to obtain combinedexpression of two ISP proteins in transgenic plants as summarized below:

I. Chimeric gene constructs whereby two ISP DNA sequences and a markergene are transferred to the plant genome as a single piece of DNA andlead to the insertion in a single locus in the genome.

Ia. The genes can be engineered in different transcriptional units eachunder control of a distinct promoter.

To express two ISP proteins and a marker protein as separatetranscriptional units, several promoter fragments directing expressionin plant cells can be used as described above. All combinations of thepromoters mentioned above in the chimaeric constructs for one ISP geneare possible. The ISP coding region in each chimeric gene of thisinvention can be the intact isp gene or preferably aninsecticidally-effective part of the intact isp gene. The individualchimerc genes are cloned in the same plasmid vector according tostandard procedures (e.g., EP 0 193 259).

Ib. Two genes (e.g., either an isp and a marker gene or two isp genes)or more can be combined in the same transcriptional unit.

To express two isp genes in the same transcriptional unit, the followingcases can be distinguished:

In a first case, hybrid genes in which the coding region of one gene isfused in frame with the coding region of another gene can be placedunder the control of a single promoter. Fusions can be made betweeneither an isp and a marker gene or between two isp genes.

Also, between each gene encoding an ISP or an insecticidally-effectivefragment thereof, a gene fragment encoding a protease (e.g., trypsin,cysteine or serine protease)-sensitive protein part could be included,such as a gene fragment encoding a part of the ISPs which is removed orcleaved upon activation by the midgut enzymes of the target insectspecies. Alternatively, between each gene encoding an ISP or aninsecticidally-effective fragment thereof, a gene fragment can beincluded encoding a peptide of about 16–20 amino acids which has thecapability to mediate cleavage at its own C-terminus by anenzyme-independent reaction (Halpin et al., 1999, Plant J. 17, 453–459,U.S. Pat. No. 5,846,767), or a gene fragment encoding a linker peptidesequence which allows the production of a recombinant cell withsignificant toxicity to the target insects.

In a second case, the coding regions of the two respective isp genes canbe combined in dicistronic units placed under the control of a promoter.The coding regions of the two isp genes are placed after each other withan intergenic sequence of defined length. A single messenger RNAmolecule is generated, leading to the translation into two separate geneproducts. Based on a modified scanning model (Kozak, 1987, Mol. Cell.Biol. 7, 3438–3445), the concept of reinitiation of translation has beenaccepted provided that a termination codon in frame with the upstreamATG precedes the downstream ATG. Experimental data also demonstratedthat the plant translational machinery is able to synthesize severalpolypeptides from a polycistronic mRNA (Angenon et al., 1989, Mol. CellBiol. 9, 5676–5684).

Based on the mechanism of internal initiation of translation (Jacksonand Kaminski, 1995, RNA 1, 985–1000) initiation of translation of thesecond gene occurs by binding of the 43S pre-initiation complex to aspecific intergenic sequence (internal ribosome entry sequence; IRES).Experimental data also demonstrated that the plant translationalmachinery is able to synthesize several polypeptides from apolycistronic mRNA containing intergenic IRES-elements (Hefferon et al.,1997, J Gen Virol 78, 3051–3059; Skulachev at al., 1999, Virology 263,139–154; PCT patent publication WO 98/54342).

II. A chimeric construct with one isp gene that is transferred to thegenome of a plant already transformed with one isp gene:

Several genes can be introduced into a plant cell during sequentialtransformation steps (retransformation), provided that an alternativesystem to select transformants is available for the second round oftransformation, or provided that the selectable marker gene is excisedfrom the plant genome using DNA recombination technology (e.g.,published PCT applications WO 94/17176 and WO 91/09957). Thisretransformation leads to the combined expression of isp genes which areintroduced at multiple loci in the genome. Preferably, two differentselectable marker genes are used in the two consecutive transformationsteps. The first marker is used for selection of transformed cells inthe first transformation, while the second marker is used for selectionof transformants in the second round of transformation. Sequentialtransformation steps using kanamycin and hygromycin have been described,for example by Sandier et al. (1988, Plant Mol. Biol. 11, 301–310) andDelauney et al. (1988, Proc. Natl. Acad. Sci. U.S.A. 85, 4300–4304).

III. Chimeric constructs with isp genes, that are separately transferredto the nuclear genome of separate plants in independent transformationevents and are subsequently combined in a single plant genome throughcrosses.

The first plant should be a plant transformed with a first isp gene oran F1 plant derived thereof through selfing (preferably an F1 plantwhich is homozygous for the isp gene). The second plant should be aplant transformed with a second isp gene or an F1 plant derived thereofthrough selfing (preferably an F1 plant which is homozygous for thesecond isp gene). Selection methods can be applied to the plantsobtained from this cross in order to select those plants having the twoisp genes present in their genome (e.g., Southern blotting) andexpressing the two ISPs (e.g., separate ELISA detection of theimmunologically different ISPs). This is a useful strategy to producehybrid varieties from two parental lines, each transformed with adifferent isp gene, as well as to produce inbred lines containing twodifferent isp genes through crossing of two independent transformants(or their F1 selfed offspring) from the same inbred line.

IV. Chimeric constructs with one or more isp genes separatelytransferred to the genome of a single plant in the same transformationexperiment leading to the insertion of the respective chimeric genes atthe same or at multiple loci.

Cotransformation involves the simultaneous transformation of a plantwith two different expression vectors, one containing a first isp gene,the second containing a second isp gene. Along with each isp gene, adifferent marker gene can be used, and selection can be made with thetwo markers simultaneously. Alternatively, a single marker can be used,and a sufficiently large number of selected plants can be screened inorder to find those plants having the two isp genes (e.g., by Southernblotting) and expressing the two proteins (e.g., by means of ELISA).Cotransformation with more than one T-DNA can be accomplished by usingsimultaneously two different strains of Agrobacterium, each with adifferent Ti-plasmid (Depicker et al., 1985, Mol. Gen. Genet. 201,477–484) or with one strain of Agrobacterium containing two T-DNAs onseparate plasmids (de Framond et al., 1986, Mol. Gen. Genet. 202,125–131). Direct gene transfer, using a mixture of two plasmids orfragments thereof, can also be employed to cotransform plant cells witha selectable and a non-selectable gene (Schocher et al., 1986,Bio/technology 4, 1093–1096).

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce more transformed plants with the samecharacteristics or to introduce the insecticidally effective isp gene inother varieties of the same or related plant species. Seeds, which areobtained from the transformed plants, contain the insecticidallyeffective isp gene as a stable genomic insert. Cells of the transformedplant can be cultured in a conventional manner to produce theinsecticidally effective portion of the ISP protein, which can berecovered for use in conventional insecticide compositions againstinsects. Of course, the above possibilities of combined production ofISP proteins in plants are as well applicable to active fragments of theISP proteins or the ISP equivalents of this invention. Theinsecticidally effective isp gene is inserted in a plant cell genome sothat the inserted gene is downstream (i.e., 3′) of, and operably linkedto, a promoter which can direct the expression of the gene part in theplant cell. This is preferably accomplished by inserting the ispchimeric gene in the plant cell genome, particularly in the nuclear orchloroplast genome. Preferred promoters include: the strong constitutive35S promoters (the “35S promoters”) of the cauliflower mosaic virus(CaMV) of isolates CM 1841 (Gardner et al., 1981, Nucl. Acids Res. 9,2871–2887), CabbB-S (Franck et al., 1980, Cell 21, 285–294) and CabbB-JI(Hull and Howell, 1987, Virology 86, 482–493); promoters from theubiquitin family (e.g., the maize ubiquitin promoter of Christensen etal., 1992, Plant Mol. Biol. 18, 675–689; see also Cornejo et al., 1993,Plant Mol. Biol. 23, 567–581), the gos2 promoter (de Pater et al., 1992,Plant J. 2, 837–844), the emu promoter (Last et al., 1990, Theor. Appl.Genet. 81, 581–588), rice actin promoters such as the promoter describedby Zhang et al. (1991, The Plant Cell 3, 1155–1165); and the TR1′promoter and the TR2′ promoter (the “TR1′promoter” and “TR2′ promoter”,respectively) which drive the expression of the 1′ and 2′ genes,respectively, of the T-DNA (Velten et al., 1984, EMBO J 3, 2723–2730).Alternatively, a promoter can be utilized which is not constitutive butrather is specific for one or more tissues or organs of the plant (e.g.,leaves and/or roots) whereby the inserted isp gene is expressed only incells of the specific tissue(s) or organ(s). In a preferred embodimentof the invention, the insecticidally effective isp gene is selectivelyexpressed in the roots of a plant, preferably corn, by placing theinsecticidally effective gene part under the control of a root-preferredpromoter (i.e., a promoter which is most active in root tissue,preferably a promoter with no or little transcription in non-root tissuesuch as pollen, leaves and stem, more particularly a promoter onlyresulting in detectable transcription in root tissue). Root-preferredpromoters need not be exclusively active in root tissue. Root-preferredpromoters in accordance with this invention include but are not limitedto: promoters located immediately upstream of a DNA sequencecorresponding to a root-specific cDNA, preferably from corn; thepromoter of U.S. Pat. No. 5,837,876, published PCT patent applicationsWO 00/29594, WO 00/73474, WO 01/00833, or U.S. Pat. No. 6,008,436; theZRP2 promoter of U.S. Pat. No. 5,633,363 and Held et al. (1997, PlantMol. Biol. 35, 367–375, Genbank accession number U38790); the promoterof U.S. Pat. No. 5,817,502; the promoter described by de Framond (1991,FEBS 290: 103–106; EP 0 452 269), the root-specific promoter of theperoxidase gene POX1 from wheat (Hertig et al., 1991, Plant Molec. Biol.16, 171–174), the promoter of U.S. Pat. No. 5,837,848, the promoter ofPCT publication WO 015662, the promoter of Goddemeier et al. (1998,Plant Mol. Biol. 36, 799–802). Other root-specific or root-preferredpromoters which may be useful in this invention include the promotersdescribed by Hirel et al., 1992, Plant Mol. Biol. 20, 207; Keller andBaumgartner, 1991, The Plant Cell 3, 1051–1061; Sanger et al., 1990,Plant Mol. Biol. 14, 433–443; Miao et al., 1991, The Plant Cell 3,11–22; Bogusz et al., 1990, The Plant Cell, 2, 633–641; Leach andAoyagi, 1991, Plant Science 79, 69–76; Teeri et al., 1989, EMBO Journal8, 343–350, all of which are incorporated herein by reference.

Expression in leaves of a plant can be achieved by using the promoter ofthe ribulose-1,5-bisphosphate carboxylase small subunit gene of theplant itself or of another plant such as pea as disclosed in U.S. Pat.No. 5,254,799. Another alternative is to use a promoter whose expressionis inducible (e.g., by temperature or chemical factors). Also, for cornplants, particularly at the time adult corn rootworms are present incorn rootworm fields, expression in pollen is preferred, by usingpromoter regions resulting in expression in corn pollen, e.g. thepromoters described in published PCT applications WO 93/25695 and WO01/12799.

The insecticidally effective isp gene is inserted in the plant genome sothat the inserted gene part is upstream (i.e., 5′) of suitable 3′ endtranscription regulation signals (i.e., transcript formation andpolyadenylation signals).

This is preferably accomplished by inserting the isp chimeric gene inthe plant cell genome. The choice of the 3′ regulatory sequence in thechimeric gene of the invention is not critical. In some cases goodexpression can be obtained when no such region is present in thechimeric gene, since a DNA sequence at the insertion site can act as a3′ regulatory sequence. Preferred polyadenylation and transcriptformation signals include those of the CaMV 35S (Mogen et al., 1990, ThePlant Cell 2, 1261–1272), the octopine synthase gene (Gielen et al.,1984, EMBO J 3, 835–845), the nopaline synthase gene (Depicker et al.,1982, J. Mol. Appl. Genet. 1, p. 561), and the T-DNA gene 7 (Velten andSchell, 1985, Nucleic Acids Research 13, 6981–6998), which act as3′-untranslated DNA sequences in transformed plant cells.

The insecticidally effective isp gene can optionally be inserted in theplant genome as a hybrid gene (U.S. Pat. No. 5,254,799; Vaeck et al.,1987, Nature 327, 33–37) under the control of the same promoter as aselectable marker gene, such as the neo gene (EP 0 242 236) encodingkanamycin resistance, so that the plant expresses a fusion protein.

All or part of the isp gene, can also be used to transform otherbacteria, such as a B. thuringiensis strain which has insecticidalactivity against Lepidoptera or Coleoptera, or root-colonizing bacteria,e.g. root-colonizing Pseudomonas putida (Vilchez et al., J. Bacteriol.182, 91–99). Thereby, a transformed bacterial strain can be producedwhich is useful for combatting insects and which can affect a widespectrum of lepidopteran and coleopteran insect pests. Transformation ofbacteria, such as bacteria of the genus Agrobacterium, Bacillus orEscherichia, with all or part of the isp gene of this invention or itsequivalent, incorporated in a suitable cloning vehicle, can be carriedout in a conventional manner, such as heat shock transformation(Bergmans et al., 1981, J. Bacteriol 146, 564–570) or conventionalelectroporation techniques as described in Mahillon et al. (1989, FEMSMicrobiol. Letters 60, 205–210) and in PCT Patent publication WO90/06999.

Transformed bacterial strains, such as Bacillus species strains,preferably Bacillus thuringiensis strains, containing the isp gene ofthis invention can be fermented by conventional methods (Dulmage, 1981,“Production of Bacteria for Biological Control of Insects” in BiologicalControl in Crop Production, Ed. Paparizas, D. C., Osmun Publishers,Totowa, N.J., USA, pp. 129–141; Bernhard and Utz, 1993, “Production ofBacillus thuringiensis insecticides for experimental and commercialuses”, In Bacillus thuringiensis, An Environmental Biopesticide: Theoryand Practice, pp. 255–267, eds. Entwistle, P. F., Cory, J. S., Bailey,M. J. and Higgs, S., John Wiley and Sons, New York) to provide highyields of cells.

An insecticidal, particularly anti-coleopteran, preferably anti-cornrootworm composition of this invention can be formulated in aconventional manner using the microorganisms transformed with the ispgene, or preferably their respective ISP proteins or insecticidallyeffective portions thereof as an active ingredient, together withsuitable carriers, diluents, emulsifiers and/or dispersants (e.g., asdescribed by Bernhard and Utz, 1993, supra). This insecticidecomposition can be formulated as a wettable powder, pellets, granules ordust or as a liquid formulation with aqueous or non-aqueous solvents asa foam, gel, suspension, concentrate, etc. Known microorganisms includecells of Pseudomonas or other bacteria that serve to encapsulate theproteins in a stable environment prior to application to the insects.Also included in the invention is a product comprising the ISP1A andISP2A protein of the invention as a combined preparation forsimultaneous, separate or sequential use to protect corn plants againstcorn rootworms, particularly such product is an insecticidal compositionor a transgenic corn plant.

A method for controlling insects, particularly Coleoptera, in accordancewith this invention can comprise applying (e.g., spraying), to a locus(area) to be protected, an insecticidal amount of the ISP proteins orhost cells transformed with the isp gene of this invention. The locus tobe protected can include, for example, the habitat of the insect pestsor growing vegetation or an area where vegetation is to be grown.

To obtain the ISP protein, cells of the recombinant hosts expressing theISP protein can be grown in a conventional manner on a suitable culturemedium and the protein can then be obtained from the medium usingconventional means. The ISP protein can then be separated and purifiedby standard techniques such as chromatography, extraction,electrophoresis, or the like. The protease-resistant toxin form can thenbe obtained by protease, e.g. cysteine or serine protease, digestion ofthe protein.

Bio-assays for testing the insecticidal efficacy are well known in theart. A method for corn rootworm testing is described in Marrone et al.(1985, J. Econ. Entomol. 78, 290–293), but many other methods areavailable to test insects on artificial diet or on transgenic plants,e.g. U.S. Pat. No. 5,990,383. In a suitable bio-assay the propercontrols are included to check for background mortality.

The following Examples illustrate the invention, and are not provided tolimit the invention or the protection sought. Many variants orequivalents of these Examples can be made or designed by a person ofordinary skill in the art without departing from the teachings of theinvention and the common knowledge. The sequence listing referred to inthis application (which forms part of this application and is attachedthereto), is as follows:

Sequence Listing:

-   SEQ ID No. 1—amino acid and DNA sequence of ISP1A protein and DNA-   SEQ ID No. 2—amino acid sequence of ISP1A protein.-   SEQ ID No. 3—amino acid and DNA sequence of ISP2A protein and DNA.-   SEQ ID No. 4—amino acid sequence ISP2A protein.-   SEQ ID No. 5—DNA sequence of the short zrp2 promoter fragment (n=any    nucleotide)-   SEQ ID No. 6—DNA sequence of the long zrp2 promoter fragment (n=any    nucleotide)-   SEQ ID No. 7—DNA sequence encoding ISP2A-1 protein-   SEQ ID No. 8—amino acid sequence of ISP2A-1 protein-   SEQ ID No. 9—DNA sequence encoding ISP1A-1 protein fragment-   SEQ ID No. 10—amino acid sequence of ISP1A-1 protein

Unless otherwise stated in the Examples, all procedures for making andmanipulating recombinant DNA are carried out by the standard proceduresdescribed in Sambrook et al., Molecular Cloning—A Laboratory Manual,Second Ed., Cold Spring Harbor Laboratory Press, NY (1989), and inVolumes 1 and 2 of Ausubel et al. (1994) Current Protocols in MolecularBiology, Current Protocols, USA. Standard materials and methods forplant molecular biology work are described in Plant Molecular BiologyLabfax (1993) by R. R. D. Croy, jointly published by BIOS ScientificPublications Ltd (UK) and Blackwell Scientific Publications (UK).Procedures for PCR technology can be found in “PCR protocols: a guide tomethods and applications”, Edited by M. A. Innis, D. H. Gelfand, J. J.Sninsky and T. J. White (Academic Press, Inc., 1990).

EXAMPLES Example 1 Characterization of the IB120-A Strain

A bacterial strain, named herein the IB120-A strain, was found in afield in the state of Morelos, Mexico.

The strain was grown overnight on LB agar plates (LB medium with 1.5%agar added; LB medium:10 g/l trypton, 10 g/l NaCl, 5 g/l yeast extract,pH 7.3) at 28° C. For small-scale cultures, 20 ml TB medium (TerrificBroth: 12 g/l tryptone, 24 g/l yeast extract, 3.8 g/l KH₂PO₄, 12.5 g/lK₂HPO₄, 5 ml/l glycerol, pH 7.1) was inoculated and grown for 65 hoursat 28° C. on a rotating platform having about 70 rotations per minute.After 65 hours, a protease inhibitor mixture was added to the culture.This cocktail has the following ingredients (volumes given are thoserequired to add to one 20 ml culture): 200 μl PMSF (100 mM), 200 μl of amixture of benzamidine.HCl (100 mM) and epsilon-amino-n-caproic acid(500 mM), 400 μl EGTA (0.5M), 40 μl antipain (0.5 mg/ml)/leupeptin (0.5mg/ml) and 20 μl beta-mercapto ethanol (14M). The culture medium, towhich the protease inhibitor mixture had been added, was thencentrifuged for 20 minutes at 3000 rpm. In some cases, the supernatantwas concentrated about 4 times using Centriprep YM-10 Centrifugal FilterDevices (Millipore Cat. No. 4305). For long term storage, a loop ofsporulated cells was added to 0.5 ml of 25% or 50% glycerol and aftervortexing, stored at −70° C. Sporulated cells were obtained by growth ofthe strain on LB agar plates until sporulation (as apparent under thelight microscope).

After cultivating on LB agar plates of single cell colonies,microscopical analysis of the IB120-A strain culture showed the presenceof rod-shaped motile single vegetative cells and swollen sporangiacontaining an ovale spore. No parasporal crystals were detected incultures of the IB120-A strain.

Supernatant of the IB120-A strain showed strong toxicity to Diabroticavirgifera larvae (hereinafter termed “Dv”) in surface contaminationassays on artificial diet (diet as described by Sutter et al. (1971, J.Econ. Entomol. 64, 65–67)). The assays were done at 24° C. (+/−1° C.) in24 multiwell Costar plates at 50-microliter toxin solution per well (2cm²), 6 wells were analyzed with 4 L1 (first instar) larvae per well foreach sample (scoring after 7 days).

Screening of supernatant harvested at 7, 24, 30, 48 and 120 hours afterinitiation of the culture showed the strongest toxicity to Dv at 48hours, with no significant toxicity found at 7 hours. At 48 hours,mortality in the 50–60% range was still found when the supernatant (attotal protein concentration of 214 microgr/ml) was diluted at 1/1024.

Loss of activity of IB120-A supernatant harvested 48, 65 and 144 hoursafter culture initiation (in dilutions of 1/1 to 1/8) upon heattreatment and retention of activity after ammonium sulphateprecipitation indicated that the toxic compound is likely a protein.

Preliminary characterization of the IB120-A strain by PCR primers forgyrase B genes (Yamada et al., 1999, Appl. Environm. Microbiol. 65,1483–1490) suggested that it was not a Bacillus strain of the subspeciesthuringiensis, anthracis or cereus. Also, no amplification products wereobtained using PCR primers specifically characterizing the 16S RNAsequences of the genus Paenibacillus which are also recognizing thespecies Bacillus lentimorbus and Bacillus popilliae (Pettersson et al.,1999, Int J Syst Bacteriol. 49(2), 531–540). Growth in NB (nutrientbroth: 3 g/l bacto beef extract, 5 g/l bacto peptone, pH 6.8) mediumindicated that the new strain was not a Bacillus larvae species. Thus,this IB120-A strain seems to be different from previously isolatedBacillus strains known to produce secreted insecticidal proteins thatare not contained in crystals.

Based on the rod-like shape and the aerobic growth, the strain could bea Bacillus species strain. With a set of general and specific primersfor cry3 Bacillus thuringiensis genes, no amplification products werefound when analyzing strain IB120-A.

Detailed analysis of the IB 120-A strain using standard microbialidentification techniques, including fatty acid analysis and API 50CHBtests combined with API 20E tests, showed that this strain is aBrevibacillus laterosporus species strain.

Example 2 Isolation and Characterization of isp1 and isp2 DNAs/Proteins

In order to isolate the genes responsible for the toxicity of IB120-A,total DNA from this strain was prepared and partially digested withSau3A. The digested DNA was size fractionated on a sucrose gradient andfragments ranging from 7 kb to 10 kb were ligated to the BamH1-digestedand TsAP (thermosensitive alkaline phosphatase)-treated cloning vectorpUC19 (Yannisch-Perron et al, 1985, Gene 33, 103–119.). The ligationmixture was electroporated in E. coli XL1-Blue cells. Transformants wereplated on LB-triacillin plates containing Xgal and IPTG and whitecolonies were selected to be used in filter hybridization experiments.Recombinant E. coli clones containing the vector were then screened witha DIG labeled probe, which was prepared as follows. First, a PCR wasperformed using as template cells from strain IB120-A. The resultingamplification product was gel-purified and used as template in asecondary PCR reaction using digoxigennin (“DIG”)-labeled dNTPs and thesame primers as in the first PCR reaction. An appropriate amount of thisamplification product was used in hybridization reactions.

Following the identification of a positive colony containing a plasmidharboring the full length isp genes, the sequence of these genes wasdetermined using the dye terminator labeling method and a Perkin ElmerABI Prism-377 DNA sequencer. The sequences of the 2 open reading framesfound in the cloned DNA fragment of a positive colony are shown in SEQID No. 1 and 3. These DNA sequences were found to be organized in anoperon and encode novel proteins, ISP1A (SEQ ID No. 2) and ISP2A (SEQ IDNo. 4), which have been found to be the causal agents of the highinsecticidal activity observed. A positive colony containing thepUC-derived plasmid with the genes responsible for toxicity (in plasmidpUCIB120/ISP) has been deposited under the provisions of the Budapesttreaty in E. coli XL1Blue as LMBP 4009 on Jan. 11, 2000.

A plasmid preparation was made from the positive colony and this plasmidwas cut using XbaI and EcoRI (resulting in an about 7 Kb fragment) andligated in the shuttle vector pSL40 that had been cut using the samerestriction enzymes. This yielded plasmid pSLIB120/ISP. The plasmidpSLIB120/ISP was then transferred into a crystal-minus B. thuringiensisstrain. Supernatant from this recombinant Bt strain was obtained asfollows.

The strain was grown overnight on LB agar plates containing erythromycin(20 μg/ml) at 28° C. For small-scale cultures, 20 ml TB mediumcontaining erythromycin (20 μg/ml) was inoculated and grown for 65 hoursat 28° C. on a rotating platform having about 70 rotations per minute.After 65 hours, a protease inhibitor mixture was added to the culture.This cocktail has the following ingredients (volumes given are thoserequired to add to one 20 ml culture): 200 μl PMSF (100 mM), 200 μl of amixture of benzamidine.HCl (100 mM)/epsilon-amino-n-caproic acid (500mM), 400 μl EGTA (0.5M), 40 μl antipain (0.5 mg/ml)/leupeptin (0.5mg/ml) and 20 μl beta-mercapto ethanol (14M). The culture medium, towhich the protease inhibitor mixture had been added, was thencentrifuged for 20 minutes at 3000 rpm. In some cases, the supernatantwas concentrated about 4 times using centriprep YM-10 Centrifugal FilterDevices (Millipore, Cat. No. 4305).

Insecticidal activity of the supernatant at 48 hours after cultureinitiation of the recombinant Bt strain containing the pSLIB120/ISPplasmid using the Dv surface contamination assay described above, showedthat the supernatant still had significant mortality in the 60% range ata 1/1024 dilution, while control mortality (the controls includedsupernatant of the untransformed Bt strain) was 0%. This shows that theisolated DNA encodes an insecticidal ingredient of the IB120-A strain,and upon transfer to another bacterium is also expressed and secreted inthe culture medium. Analysis of toxicity of another independentBt-crystal-minus strain transformed with the pSLIB120/ISP plasmidconfirmed the high insecticidal activity of the supernatants compared tothat of the untransformed control.

The LC50 value of the supernatant of the Bt strain expressing the twoISP proteins was found to be 3.5 ng/cm² after 4 days using theabove-described surface contamination assay with Dv larvae (based ontotal supernatant protein concentration). Detailed analysis of thetoxicity of supernatant produced by the recombinant Bt strain expressingthe ISP1A and ISP2A proteins to selected coleopteran insects is reportedin Table 1 below. The ISP proteins of the invention were found to havesignificant insecticidal activity to Western, Northern and Southern cornrootworm larvae, and also to larvae of the Colorado potato beetle andthe cotton boll weevil.

The mature ISP1 and ISP2 proteins were purified to apparent homogeneityby ammonium sulphate precipitation followed by passage over differentchromatographical columns. The chromatographical analyses suggest thatthe ISP1A and ISP2A proteins are present in the supernatant in equimolarratio. The ISP1A protein was found to be of rather hydrophobic nature,while ISP2A was of rather hydrophilic nature. Amino-terminal sequencedetermination of the mature ISP1A and ISP2A proteins produced in therecombinant crystal-minus Bt strain showed that amino acid position 38in SEQ ID No. 2 for ISP1A and amino acid position 51 in SEQ ID No. 4 forISP2A are the N-terminal amino acids of the active proteins present insupernatant of such transformed Bt strain. The N-terminus for ISP1A wasfound to be IATTTQASKD (position 38 to 47 of SEQ ID No: 2), and that forISP2A was found to be LVKTTNNTED (position 51 to 60 of SEQ ID No: 2,which matches fully with the amino acid sequence of the DNA sequencesisolated.

The apparent molecular weight of the pure mature ISP1A protein wasdetermined to be about 100 kD in 8% SDS-PAGE gel electrophoresis, thatof the pure mature ISP2A protein was determined to be about 45 kDprotein in 10% SDS-PAGE gel electrophoresis, using molecular weightmarkers of 37, 50, 75, 100, and 150 kD.

The activity of the mature ISP1A and ISP2A proteins as produced by therecombinant strain was evaluated against several insects. The resultsagainst selected coleopteran insects are shown in Table 1 below.

TABLE 1 Colepteran activity of ISP1A-ISP2A: LC50 (μg/ml) LC90 (μg/ml)Insect Stage (95% CL) (95% CL) Dv L1 0.437 1.689 (0.321–0.563)(1.250–2.597) L2 3.84 117.4 (−) (−) L3 21.674* 531.76 (5.012–100.432)(110.688–86864) Db L1 0.213 0.890 (0.116–0.338) (0.542–2.006) Du L1 4.9130.06 (1.65–13.26) (11.52–329.72) Ld L1 + 2d 0.037 1.068 (−) (−) Ag L1207.1 8759.2 (84.3–654.7) (1865.8–620175.8)Legend to Table 1:

Dv: Diabrotica virgifera, Western corn rootworm; Db: Diabrotica barber,Northern corn rootworm; Du: Diabrotica undecimpunctata howardi, Southerncorn rootworm; Ld: Leptinotarsa decemlineata, Colorado potato beetle;Ag: Anthonomus grandis, cotton boll weevil; L1: first larval stage; L2:second larval stage; L3: third larval stage; L1+2d: 2d after egg hatch;*: 90% CL (CL=Confidence Limits, LC50: total supernatant proteinconcentration when 50% of the insects are killed).

Bioassays used: Dv, Db, Du: surface contamination assay on artificialdiet (see above for description) at ratio of 25 μl/cm², scoring after 7days; Ld: diptest with potato foliage (cut potato foliage dipped intoxin solution, allowed to dry and put in a petri dish (9 cm diam.) with10 larvae; after 1 day untreated foliage was added to the petridish,scoring was after 5 days); Ag: artificial diet incorporation test: dietas described by Moore and Whisnant, Handbook of Insect Rearing, Vol. I,Pritah Singh and R. F. Moore; Elsevier, Amsterdam, 1985, p. 217; assay:incorporation of 2 ml toxin solution per 25 ml diet, in 24 multiwellCostar plates (about 1 ml/well), 1 egg per well, 20 wells per sample,scoring after 14 days).

In the assays reported in Table 1, controls (untreated food, foodsupplied with supernatant of a non-transformed Bt-crystal-minus strainor with water) did not show any mortality above 20% for any of the aboveinsects (a variation from 5 to 20% control mortality was found accordingto larval stage and insect species (the 20% value is for the thirdlarval stage of Dv)).

The LC50 values obtained for Diabrotica virgifera larvae using thebio-assay as described above, when an equimolar combination of ISP1A andISP2A protein was applied after purification of these proteins from thesupernatans of the recombinant Bt strain, were not significantlydifferent from those obtained above.

Bio-assays of a mixture of the mature ISP1A and ISP2A protein instandard surface contamination assays against selected lepidopteraninsects (Ostrinia nubilialis, Heliothis virescens, Helicoverpa zea,Spodoptera frugiperda, Sesamia nonagrioides, Spodoptera littoralis,Helicoverpa armigera, and Manduca sexta) showed that the ISP proteins ofthis invention do not cause any mortality in these insects above controllevels. This evidences the coleopteran-specific nature of the proteinsof this invention. Preliminary bio-assays with two aphid species alsoshowed that a mixture of the ISP1A and ISP2A proteins did not cause anysignificant mortality in these insects.

To determine which fragment of the isp genes are sufficient to encode anISP protein complex toxic to Diabrotica larvae, a series of C-terminallytruncated ISP proteins were made by inserting a stopcodon in the ORF ofthe isp genes at different positions (and thus making different 3′ genetruncations). The stopcodons were inserted at random positions using theGenome Priming System (GPS, New England Biolabs, catalog #7100). Usingthis system, a 1.7 kb transposon (containing stopcodons in all threereading frames and 2 sequences complementary to specific primers) isinserted randomly but only once in the ‘target’ DNA. The position of theinsertion, and therefore, the truncation can then be estimated by PCRscreening using a gene-specific primer and a transposon-specific primeror can be precisely determined by sequencing. A set of constructs with atransposon at different positions in one of the isp genes was thentransformed individually in a crystal minus Bt strain and thesusceptibility of Western corn rootworm larvae to the supernatant ofeach recombinant Bt strain was tested using the assay described above.

For truncation of the isp2 gene, a fragment containing the gene was cutout of pSLIB120/ISP using BstBI and PmII. Following the GPS reaction,this fragment was then ligated into the pSLIB120/ISP vector, cut withthe same enzymes. Recombinant E. coli colonies were PCR-screened inorder to estimate the position of the transposon insertion site. A setof 10 clones with the transposon at different positions in the secondhalf (3′ half) of the isp2 gene was selected. Plasmid DNA of theseclones was transformed in a dcm and dam methylase negative E. colistrain GM2163. Plasmid DNA isolated from this strain was thentransformed in a crystal-minus Bt strain. Supernatant was prepared andbioassayed, using supernatant from the Bt strain transformed withpSLIB120/ISP as a positive control and supernatant from thecrystal-minus Bt strain as a negative control. For truncation of theisp1 gene, a fragment was cut out of pSLIB120/SP using PmII and EcoRI.The same methodology was followed as for the truncation of the isp2gene.

The results of the transposon insertion analysis indicate thattruncation of relatively large segments of the isp2A gene at the 3′ endabolishes toxicity, indicating that no or only a relatively shortC-terminal truncation is allowed in the ISP2A protein. After sequencing,the smallest C-terminally truncated ISP2A toxin fragment retainingtoxicity in this study when tested in combination with the ISP1A proteinwas found to end at amino acid position 449 in SEQ ID No. 4.

The results of these analyses indicate that a larger truncation of theISP1A protein at the 3′ end is possible: sequence determination showedthat the toxic fragment with the largest C-terminal truncation ended atamino acid position 768 in SEQ ID No. 2. Thus, approximately 300 bp ofthe ISP1A coding region can be deleted at the 3′ side without losingtoxicity.

The above study also indicates that insecticidal activity is reduced tothe background level when no functional ISP1A is produced, confirmingthe binary nature of the ISP proteins of this invention.

The ISP2A protein from amino acid position 51 to amino acid position 449in SEQ ID No. 4 and the ISP1A protein from amino acid position 38 toamino acid position 768 in SEQ ID No. 2 are thus useful insecticidalfragments of the ISP proteins.

Furthermore, a chimeric gene containing a DNA fusion of the ISP1A andISP2A genes was expressed in a crystal-minus Bacillus thuringiensisberliner 1715 strain. A DNA sequence encoding an Arg-Lys-linker (RKRKRK)(SEQ ID NO: 11) or a DNA sequence encoding a Gly-linker (GGGGGG) (SEQ IDNO: 12) was provided between the open reading frames encoding the ISP2Aand the ISP1A proteins (ISP2A still has its N-terminal signal peptidepresent, while ISP1A lacks its signal peptide), so that a translationalfusion protein is produced; i.e. a fusion protein in which the speficiedlinker connects the ISP2A protein (as the N-terminal fusion partner) tothe ISP1A protein (as the C-terminal fusion partner). The recombinant Btstrain proved to cause mortality to Diabrotica virgifera that wassignificantly higher then that of the untransformed control strain(i.e., the crystal-minus Bt 1715 strain). This shows the possibility ofproducing the ISP proteins of the invention as a fusion protein in arecombinant host cell by using a single chimeric gene. Obviously, asexplained in the description of the invention, multiple differentapproaches can be used to achieve the production of a fusion protein inthe recombinant cell, e.g. cells of a plant.

Example 3 Production of ISP Proteins in Plants

To obtain optimal expression in plants, the isp1A and isp2A native DNAsequences are modified to produce modified DNA sequences encoding theISP1A and ISP2A proteins. The optimized DNA sequences encoding the ISP1A-1 and ISP2A-1 proteins with their signal peptides replaced by amethionine and alanine amino acid are shown in SEQ ID No. 9 and 7,respectively.

These coding regions are inserted in a chimeric gene operably linked tosuitable regulatory sequences, e.g., a promoter based on the short orlong zrp2 promoter sequences of SEQ ID No. 5 or 6 using standardtechniques, and appropriate 3′ transcription termination andpolyadenylation sequences. In some constructs, the above chimeric genescontain a DNA sequence encoding the optimized chloroplast transitpeptide (in place of the N-terminal bacterial signal peptide) asdescribed in U.S. Pat. 5,510,471 (or Reissued U.S. RE Pat. No. 036449)resulting in targeting to the plastids or a signal peptide causingtargeting to the cell wall. Other signal peptides effective in plantcells can similarly be used, preferably the signal peptide encoded bythe alpha-amylase 3 gene of rice (Sutliff et al., 1991, Plant Molec.Biol. 16, 579–591), the signal peptide encoded by the ferredoxin NADP+oxidoreductase gene from spinach (Oelmueller et al., 1993, Mol. Gen.Genet. 237, 261–272), or the signal peptide encoded by the proteinaseinhibitor II gene of potato (Keil et al., 1986, Nucl. Acids Res. 14,5641–5650).

Corn cells are stably transformed with the above isp1A and isp2Achimeric genes using Agrobacterium-mediated transformation (Ishida etal., 1996, Nature Biotechnology 14, 745–750; and U.S. Pat. No.5,591,616), protoplast transformation as described in U.S. Pat. No.5,792,936, or by electroporation using wounded and enzyme-degradedembryogenic callus, as described in WO 92/09696 or U.S. Pat. No.5,641,664 (all of these above references are incorporated herein byreference). Corn plants are regenerated from the transformed cells andare selected for optimal expression levels in roots and for adequateoverall agronomic characteristics. The thus obtained transgenic cornplants producing the above ISP proteins of the invention causesignificant mortality to Diabrotica virgifera larvae attempting to feedon such plants.

In accordance with this invention, stably transformed corn plants arealso obtained containing several DNA constructs, so that the corn plantcan express one or both the ISP proteins of the invention in its cellsunder control of the suitable regulatory control regions, together withan appropriate marker gene, preferably a herbicide resistance gene. Inthese DNA constructs, a preferred 3′ transcription termination andpolyadenylation sequence is a 215 bp fragment containing the 3′transcription termination and polyadenylation sequences obtained fromCauliflower Mosaic virus (Oka et al., 1981, J. Mol. Biol. 147, 217–226).In some constructs, a 5′ untranslated leader sequence can be added,preferably those chosen from the leader sequences of a cab22 gene fromPetunia (Harpster et al., 1988, Mol. Gen Genetics 212, 182–190) or thezrp2 gene (Held et al, 1997, Plant Molecular Biology 35, 367–375,Genbank accession number U38790). Preferred promoters in the constructsare promoter-effective parts of the 35S promoter (e.g., Odell et al.,1985, Nature 313, 810–812) or zrp2 promoters (Held et al, 1997, PlantMolecular Biology 35, 367–375, Genbank accession number U38790). Theconstructs can also contain a signal peptide effective in plant cellsbesides the above optimized chloroplast transit peptide, namely thesignal peptide of the alpha-amylase 3 gene of rice (Sutiff et al., 1991,Plant Molec. Biol. 16, 579–591), the signal peptide of the ferredoxinNADP+ oxidoreductase from spinach (Oelmueller et al., 1993, Mol. Gen.Genet. 237, 261–272), or the signal peptide from the proteinaseinhibitor II of potato (Keil et al., 1986, Nucl. Acids Res. 14,5641–5650). Transformations of plants with the ISPA chimeric genes usinga selection from the above preferred elements using techniques availableto the person of ordinary skill in the art can be used to achieve thedesired goal of the invention.

Preferably the plants also contain a gene encoding a protein conferringresistance to glufosinate or glyphosate, for which a different promoterand/or leader sequence is used, e.g. the gos2 promoter and/or the gos2leader (de Pater et al., 1992, Plant J. 2, 837–844).

Needless to say, this invention is not limited to the above corn plants,transformation methods nor to the particular ISP proteins or DNAsequences used. Rather, the invention also includes any variant orequivalent of the ISP protein retaining insecticidal activity, such as aprotein having substantially the amino acid sequence of the ISP proteinsof the invention and having substantially the insecticidal activity ofthe ISP proteins. Also, any plant which is susceptible to damage bycoleopteran insects, particularly corn rootworms, weevils or potatobeetles, especially Diabrotica virgifera or Leptinotarsa decemlineata,preferably an insect selected from the group consisting of: Agelasticaalni, Hypera postica, Hypera brunneipennis, Haltica tombacina,Anthonomus grandis, Tenebrio molitor, Triboleum castaneum, Dicladispaarmigera, Trichispa serica, Oulema oryzae, Colaspis brunnea,Lissorhorptrus oryzophilus, Phyllotreta cruciferae, Phyllotretastriolata, Psylliodes punctulata, Entomoscelis americana, Meligethesaeneus, Ceutorynchus sp., Psylliodes chrysocephala, Phyllotretaundulata, Leptinotarsa decemlineata, Diabrotica undecimpunctataundecimpunctata, Diabrotica undecimpunctata howardi, Diabrotica barberiand Diabrotica virgifera, is included in the invention as a preferredtarget for transformation with a DNA encoding an ISP protein.

1. A plant cell, seed, or plant, each transformed with a first chimericgene comprising a plant-expressible promoter operably linked to a DNAsequence encoding a protein comprising the amino acid sequence of thesmallest active toxin of the protein of SEQ ID NO: 2, and a secondchimeric gene comprising a plant-expressible promoter operably linked toa DNA sequence encoding a protein comprising the amino acid sequence ofthe smallest active toxin of the protein of SEQ ID NO: 4, wherein saidsmallest active toxin of the protein of SEQ ID NO: 2 comprises afragment of the protein of SEQ ID NO: 2 having insecticidal activity toDiabrotica virgifera larvae when ingested by said larvae in combinationwith the protein of SEQ ID NO: 4 from amino acid position 51 to 457, andwherein said smallest active toxin of the protein of SEQ ID NO: 4comprises a fragment of the protein of SEQ ID NO: 4 having insecticidalactivity to Diabrotica virgifera larvae when ingested by said larvae incombination with the protein of SEQ ID NO: 2 from amino acid position 38to
 871. 2. A plant cell, seed, or plant, each transformed with a firstDNA sequence encoding a protein comprising the amino acid sequence ofthe smallest active toxin of the protein of SEQ ID NO: 2, and a secondDNA sequence encoding a protein comprising the amino acid sequence ofthe smallest active toxin of the protein of SEQ ID NO: 4, wherein saidsmallest active toxin of the protein of SEQ ID NO: 2 comprises afragment of the protein of SEQ ID NO: 2 having insecticidal activity toDiabrotica virgifera larvae when ingested by said larvae in combinationwith the protein of SEQ ID NO: 4 from amino acid position 51 to 457, andwherein said smallest active toxin of the protein of SEQ ID NO: 4comprises a fragment of the protein of SEQ ID NO: 4 having insecticidalactivity to Diabrotica virgifera larvae when ingested by said larvae incombination with the protein of SEQ ID NO: 2 from amino acid position 38to
 871. 3. The plant cell, seed, or plant of claim 1, which is a corncell, seed, or plant.
 4. A process for producing a plant resistant to acoleopteran insect pest, comprising transforming a plant cell with afirst chimeric gene comprising a plant-expressible promoter operablylinked to a DNA sequence encoding a protein comprising the amino acidsequence of the smallest active toxin of the protein of SEQ ID NO: 2,and a second chimeric gene comprising a plant-expressible promoteroperably linked to a DNA sequence encoding a protein comprising theamino acid sequence of the smallest active toxin of the protein of SEQID NO: 4, wherein said smallest active toxin of the protein of SEQ IDNO: 2 comprises a fragment of the protein of SEQ ID NO: 2 havinginsecticidal activity to Diabrotica virgifera larvae when ingested bysaid larvae in combination with the protein of SEQ ID NO: 4 from aminoacid position 51 to 457, and wherein said smallest active toxin of theprotein of SEQ ID NO: 4 comprises a fragment of the protein of SEQ IDNO: 4 having insecticidal activity to Diabrotica virgifera larvae wheningested by said larvae in combination with the protein of SEQ ID NO: 2from amino acid position 38 to 871; and regenerating from saidtransformed plant cell a plant resistant to said insect.
 5. A processfor controlling a coleopteran insect pest, comprising planting, sowingor growing in a field seeds or plants transformed with a first chimericgene comprising a plant-expressible promoter operably linked to a DNAsequence encoding a protein comprising the amino acid sequence of thesmallest active toxin of the protein of SEQ ID NO: 2, and a secondchimeric gene comprising a plant-expressible promoter operably linked toa DNA sequence encoding a protein comprising the amino acid sequence ofthe smallest active toxin of the protein of SEQ ID NO: 4, wherein saidsmallest active toxin of the protein of SEQ ID NO: 2 comprises afragment of the protein of SEQ ID NO: 2 having insecticidal activity toDiabrotica virgifera larvae when ingested by said larvae in combinationwith the protein of SEQ ID NO: 4 from amino acid position 51 to 457, andwherein said smallest active toxin of the protein of SEQ ID NO: 4comprises a fragment of the protein of SEQ ID NO: 4 having insecticidalactivity to Diabrotica virgifera larvae when ingested by said larvae incombination with the protein of SEQ ID NO: 2 from amino acid position 38to
 871. 6. The process of claim 5, wherein said seeds or plants are cornseeds or plants.
 7. The process of claim 4, wherein said insect isselected from the group consisting of: rootworms, weevils, potatobeetles, Diabrotica species, Anthonomus spp., Leptinotarsa spp.,Agelastica alni, Hypera postica, Hypera brunneipennis, Halticatombacina, Anthonomus grandis, Tenebrio inolitor, Triboleum castaneum,Dicladispa armigera, Trichispa serica, Oulema oryzae, Colaspis brunnea,Lissorhorptrus oryzophilus, Phyllotreta cruciferae, Phyllotretastriolata, Psylliodes punctulata, Entomoscelis americana, Meligethesaeneus, Ceutorynchus sp., Psylliodes chrysocephala, Phyllotretaundulata, Leptinotarsa decemlineata, Diabrotica undecimpunctataundecimpunctata, Diabrotica undecimpunctata howardi, Diabrotica barberi,and Diabrotica virgifera.
 8. The process of claim 5, wherein said insectis selected from the group consisting of: rootworms, weevils, potatobeetles, Diabrotica species, Anthonomus spp., Leptinotarsa spp.,Agelastica alni, Hypera postica, Hypera brunneipennis, Halticatombacina, Anthonomus grandis, Tenebrio molitor, Triboleum castaneum,Dicladispa armigera, Trichispa serica, Oulema oryzae, Colaspis brunnea,Lissorhorptrus oryzophilus, Phyllotreta cruciferae, Phyllotretastriolata, Psylliodes punctulata, Entomoscelis americana, Meligethesaeneus, Ceutorynchus sp., Psylliodes chrysocephala, Phyllotretaundulata, Leptinotarsa decemlineata, Diabrotica undecimpunctataundecimpunctata, Diabrotica undecimpunctata howardi, Diabrotica barberi,and Diabrotica virgfera.
 9. The process of claim 6, wherein said insectis selected from the group consisting of: rootworms, weevils, potatobeetles, Diabrotica species, Anthonomus spp., Leptinotarsa spp.,Agelastica alni, Hypera postica, Hypera brunnezpennis, Halticatombacina, Anthonomus grandis, Tenebrio molitor, Triboleum castaneum,Dicladispa armigera, Trichispa serica, Oulema oryzae, Colaspis brunnea,Lissorhorptrus oryzophilus, Phyllotreta cruciferae, Phyllotretastriolata, Psylliodes punctulata, Entomoscelis americana, Meligethesaeneus, Ceutorynchus sp., Psylliodes chrysocephala, Phyllotretaundulata, Leptinotarsa decemlineata, Diabrotica undecimpunctataundecimpunctata, Diabrotica undecimpunctata howardi, Diabrotica barberi,and Diabrotica virgfera.
 10. The cell, seed or plant of claim 1, whereinsaid DNA sequence comprises an artificial DNA sequence having adifferent codon usage than the naturally-occurring DNA sequence SEQ IDNO: 1 or SEQ ID NO:
 3. 11. The process of claim 4, wherein said DNAsequence comprises an artificial DNA sequence having a different codonusage than the naturally-occurring DNA sequence SEQ ID NO: 1 or SEQ IDNO:
 3. 12. The process of claim 5, wherein said DNA sequence comprisesan artificial DNA sequence having a different codon usage than thenaturally-occurring DNA sequence SEQ ID NO:
 3. 13. The plant cell, seedor plant of claim 1, wherein the DNA sequence in the first chimeric geneencodes a protein comprising the amino acid sequence of SEQ ID NO:2 fromamino acid position 38 to amino acid position 768 or
 871. 14. The cell,seed or plant of claim 1, wherein the DNA sequence in the first chimericgene encodes a protein comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:
 10. 15. The cell, seed or plant of claim 1, wherein theDNA sequence in the second chimeric gene encodes a protein comprisingthe amino acid sequence of SEQ ID NO: 4 from amino acid position 51 toamino acid position 449 or
 457. 16. The cell, seed or plant of claim 1,wherein the DNA sequence in the second chimeric gene encodes a proteincomprising the amino acid sequence of SEQ ID NO: 4, or SEQ ID NO:
 8. 17.The process of claim 5, wherein the DNA sequence in the first chimericgene encodes a protein comprising the amino acid sequence of SEQ ID NO:2from amino acid position 38 to amino acid position 768 or
 871. 18. Theprocess of claim 5, wherein the DNA sequence in the first chimeric geneencodes a protein comprising the amino acid sequence of SEQ ID NO: 2 orSEQ ID NO:
 10. 19. The process of claim 5, wherein the DNA sequence inthe second chimeric gene encodes a protein comprising the amino acidsequence of SEQ ID NO: 4 from amino acid position 51 to amino acidposition 449 or
 457. 20. The process of claim 5, wherein the DNAsequence in the second chimeric gene encodes a protein comprising theamino acid sequence of SEQ ID NO: 4, or SEQ lID NO: 8.